Summary The Hedgehog (Hh)family of secreted signaling molecules has multiple roles in the development and physiology of multicellular organisms, including humans and Drosophila. These roles depend on properly regulated expression, dispersal and transduction of Hh signals. In humans, insufficient Hh signaling leads to holoprosencephaly and excessive Hh signaling pathwayactivity can produce developmental abnormalities and gives rise to many forms of cancer. Current understanding of Hh signal transduction derives largely from molecular genetic studies using Drosophila and has mostly been found to be directly applicable to vertebrate Hh signaling. This grant uses well-studied paradigms of Hh signaling in Drosophila to study how the key transducer, Smoothened (Smo), and the key transcriptional effector, Cubitus interruptus (Ci),of Hh signaling are regulated. Prior studies indicate that the activity of Ci is regulated by phosphorylation, proteolysis and binding partners that limit its access to the nucleus. Specific aims include discerning how another key protein, Slimb, binds to Ci in a phosphorylation-dependent manner to ensure its inactivation, ubiquitination and proteolysis in the absence of Hh, whether and how Ci phosphorylation is regulated by Hh, and whether Ci phosphorylation dictates its binding partners or vice versa. Smo contacts Ci via a mutual binding partner, Costal 2 (Cos2) and must be phosphorylated at specific sites to be active. A current specific aim is to determine if Hh regulates Smo phosphorylation, either directly or by stabilizing phosphorylated Smo, whether the phosphorylated region of Smo binds directly to Cos2 or to Slimb to affect Smo activity or stability, and how phosphorylation collaborates with other Hh-induced changes in Smo to alter interactions among Smo, Cos2 and Ci to activate Ci. Methods to be used include in vitro binding and ubiquitination assays, determination of phosphorylation status in vivo by using phospho-specific antibodies, and measuring Hh signaling activity with reporter genes in animals carrying a variety of mutations and transgenes encoding Smo and Ci derivatives with altered phosphorylation sites and protein binding domains. Relevance A small number of signaling molecules mediate the communications between cells that govern the normal development and physiology of multicellular organisms, including humans and the fruitfly, Drosophila. If signaling by one such molecule, Hedgehog, is altered by an inherited or novel mutation it can lead to birth defects or cancer. This grant aims to understand how Hedgehog proteins alter cell behavior by studying this process in the model genetic organism, Drosophila, so that human Hedgehog-related diseases can be diagnosed and treated better.